This invention relates to detecting eukaryotic microorganisms.
Fungal infection, and particularly invasive systemic candidiasis, is a significant cause of morbidity and mortality in immunocompromised patients. It is important in treatment of invasive candidiasis to obtain a diagnosis early enough to permit application of effective antifungal therapy. Known approaches to premortem diagnosis of candidiasis include isolation of Candida species from blood cultures, serological tests for antibody to Candida, and direct detection of Candida antigens or Candida metabolites in serum.
L. de Repentigny et al. (1985), J. Clin. Microbiol., vol. 21(6), pages 972-79, used enzyme immunoassay and gas-liquid chromatography to compare the concentrations of three metabolic markers for candidiasis in serum samples from normal blood donors and high risk patients with and without invasive candidiasis, and concluded that the best approach to diagnosis of invasive candidiasis entails obtaining blood cultures and carrying out serial assays for mannan in serum.
B. V. Kumar et al. (1985), Infect. Immun., vol. 48(1), pages 806-12, showed that human antibodies in blood serum from patients with histoplasmosis reacted most frequently to antigens shared by three disparate fungi, Histoplasma capsulatum, Candida albicans, and Saccharomyces cerevisiae, while they reacted with low frequency to antigens specific for individual fungal species. Kumar et al. suggested that specific epitopes may be present as part of the shared antigens, and that monoclonal antibodies to, e.g., the mitochondrial fraction of C. albicans might be used for specific immunodiagnosis.
B. B. Magee et al. (1987), J. Bacteriol., vol. 169(4), pages 1639-43, found differences in patterns of restriction fragment length polymorphisms ("RFLPs") for the ribosomal DNA ("rDNA") regions from clinical isolates of various species and strains of Candida, and suggested that RFLPs may be clinically useful in biotyping various strains of C. albicans or in distinguishing various species.
L. Nelles et al. (1984), Nucleic Acids Research, vol. 12(23), pages 8749-8768, proposed an alignment of fourteen small ribosomal subunit RNA ("srRNA") sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic, and plant mitochondrial origin, on the basis of the presence of conserved features in the sequences, such as regions of theoretical secondary structure formation. They identified a number of presumed double-stranded regions ("proposed helices"), and which were present in all the aligned sequences (that is, generally conserved "universal helices"), regions which occurred only in eukaryotic srRNAs (conserved among eukaryotes; "eukaryote-specific helices"), and other regions which were found only in prokaryotic srRNAs (conserved among prokaryotes; "prokaryote-Specific helices"). Nelles et al. proposed a system for numbering the corresponding regions of the aligned sequences. For example, a presumed universal helix, numbered 19 in the Nelles alignment, is at bases having nucleotide numbers 632 through 641 (A. salina-specific nucleotide numbering) in each of six eukaryotes in the alignment. They also identified "variable areas," in which the primary as well as the secondary structure appeared to be especially variable among the srRNAs, including, for example, "area V4" (bases 642-870).
Within V4 is a long eukaryotic region (bases 642-805) having low sequence conservation. This region is entirely absent from all the other aligned srRNA sequences from archaebacterial, prokayotic, eukaryotic mitochondrial, and eukaryotic chloroplast sources.
D. E. Kohne, Canadian Pat No. 1 215 904, describes a probe which is complementary to rRNA from any bacteria but is not complementary to human cell rRNA, for detecting the presence of Mollicutes species in human cell culture and other types of bacterial cells; and a method for making the probe. Kohne uses whole Mollicutes hominis rRNA as a template for reverse transcriptase synthesis of radioactive cDNA; hybridizes the cDNA to M. hominis rRNA to ensure that the cDNA is indeed complementary to this rRNA; then the labeled cDNA is hybridized with an excess of human rRNA, and the fraction not hybridizing to human rRNA is recovered to provide the probe. Kohne also describes using this method, beginning with Trypanosoma brucei rRNA, to select a cDNA probe that is complementary to trypanosome rRNA but is not complementary to human rRNA, for detection and quantitation of trypanosomes in human samples by hybridization. Kohne also suggests comparing known nucleotide sequences of rRNA from different organisms to identify "group specific sequences similar to a specific group of organisms", and making as a probe a sequence complementary to such a sequence.